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Rapid Patterning of 1-D Collagenous Topography as anECM Protein Fibril Platform for Image CytometryNiannan Xue1, Xia Li1, Cristina Bertulli1, Zhaoying Li2, Atipat Patharagulpong1, Amine Sadok3,

Yan Yan Shery Huang2*

1 Cavendish Laboratory, University of Cambridge, Cambridge, United Kingdom, 2 Department of Engineering, University of Cambridge, Cambridge, United Kingdom,

3 The Institute of Cancer Research, London, United Kingdom

Abstract

Cellular behavior is strongly influenced by the architecture and pattern of its interfacing extracellular matrix (ECM). For anartificial culture system which could eventually benefit the translation of scientific findings into therapeutic development,the system should capture the key characteristics of a physiological microenvironment. At the same time, it should alsoenable standardized, high throughput data acquisition. Since an ECM is composed of different fibrous proteins, studyingcellular interaction with individual fibrils will be of physiological relevance. In this study, we employ near-fieldelectrospinning to create ordered patterns of collagenous fibrils of gelatin, based on an acetic acid and ethyl acetateaqueous co-solvent system. Tunable conformations of micro-fibrils were directly deposited onto soft polymeric substrates ina single step. We observe that global topographical features of straight lines, beads-on-strings, and curls are dictated bysolution conductivity; whereas the finer details such as the fiber cross-sectional profile are tuned by solution viscosity. Usingthese fibril constructs as cellular assays, we study EA.hy926 endothelial cells’ response to ROCK inhibition, because of ROCK’skey role in the regulation of cell shape. The fibril array was shown to modulate the cellular morphology towards a pre-capillary cord-like phenotype, which was otherwise not observed on a flat 2-D substrate. Further facilitated by quantitativeanalysis of morphological parameters, the fibril platform also provides better dissection in the cells’ response to a H1152ROCK inhibitor. In conclusion, the near-field electrospun fibril constructs provide a more physiologically-relevant platformcompared to a featureless 2-D surface, and simultaneously permit statistical single-cell image cytometry using conventionalmicroscopy systems. The patterning approach described here is also expected to form the basics for depositing otherprotein fibrils, seen among potential applications as culture platforms for drug screening.

Citation: Xue N, Li X, Bertulli C, Li Z, Patharagulpong A, et al. (2014) Rapid Patterning of 1-D Collagenous Topography as an ECM Protein Fibril Platform for ImageCytometry. PLoS ONE 9(4): e93590. doi:10.1371/journal.pone.0093590

Editor: Wei-Chun Chin, University of California, Merced, United States of America

Received December 9, 2013; Accepted March 5, 2014; Published April 11, 2014

Copyright: � 2014 Xue et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricteduse, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This research is funded by an Oppenheimer Research Fellowship and a Department of Engineering Starting Grant (Cambridge). X. Li is supported bythe Chinese Scholarship Council (http://en.csc.edu.cn/), C. Bertulli is supported by Fondazione Angelo Della Riccia (http://theory.fi.infn.it/casalbuoni/dellariccia/)and A. Patharagulpong is supported by the Royal Thai Scholarship Council. The funders had no role in study design, data collection and analysis, decision topublish, or preparation of the manuscript.

Competing Interests: The authors have declared that no competing interests exist.

* E-mail: [email protected]

Introduction

Cell and tissue culturing has been traditionally performed on a

flat, 2-D, featureless surface. In the last decade, extensive studies

have revealed the profound influence of the spatial and physical

properties of a substratum on cellular behavior and cell fate [1,2].

These findings undoubtedly have opened up debates on the extent

to which our present understanding, derived mainly from a 2-D

petri-dish surface, is valid in vivo, e.g [3,4]. 3-D culturing in a

collagenous hydrogel matrix, for example, is seen as a better model

system to recapitulate the in vivo extracellular matrix (ECM)

microenvironment [5–7]. However, these systems require a more

sophisticated imaging platform [8] not compatible with the

existing high throughput systems based on 2-D image acquisition

and segmentation. The variability in the gel preparation [8] may

also influence the comparability of different results. Recent

findings have demonstrated that cells interfacing with 1-D tracks

of adhesion molecules resemble the migratory behavior in 3-D.

This has opened up the possibility of designing a robust 1-D

system mimicking a 3-D context [9–11]. This concept provides an

alternative approach to investigate physiological cellular behaviors

while giving a high level of compatibility with a standardized

imaging platform.

Since ECMs are composed of fibrous protein structures [1], the

study of cellular interaction with individual fibrils will be of

physiological relevance. Each fibril not only provides the 1-D

adhesive features like the cases of ref. [9,11], but also presents the

curved contour which may be experienced by cells in vivo. To

utilize the capability of electrospinning in the efficient fabrication

of bio-polymeric fibres [12–15], we adopt near-field electrospin-

ning (NFES) [16,17] to conduct fast and continuous patterning of

gelatin fibrils. NFES has the advantage over traditional far-field

electrospinning in curtailing the inherent electric instabilities [16],

and it is employed in quest of precision control of fiber

morphology, necessary to trigger well-defined cell-substrate

interactions [18]. Various applications of NFES have recently

been demonstrated, for instance, patterning polyethylene oxide

(PEO) for integrated circuit chip insulation [16,19], piezoelectric

polymers for energy conversion [20,21], conjugated polymers for

light emitting diode [22], and carbon nanotube-PEO composites

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for stretchable electrodes [17]. To our knowledge, this is the first

application of NFES to pattern ECM protein fibers. Its potential

implementation will provide a simple, rapid and versatile

alternative to the existing micro-patterning technologies such as

laser writing and lithography [23].

In this study, ordered arrays of gelatin fibers were deposited

onto an insulating elastomer surface over an area of tens of

centimeters squared. Gelatin is chosen because it is a collagen

analog [24,25] which emulates key chemical and biological

functions of a native ECM. We focus our discussion on how the

solution properties of the NFES ‘‘ink’’ can affect the fibril

morphology, at both the local profile level, and the long-range

topography level. Also by discussing the underlying mechanisms

affecting the solution properties, we propose general strategies to

tailor-design the ECM patterns by NFES, which are believed to be

of general applicability to other protein fibril systems. Finally, we

test potential applications of the NFES gelatin fiber array as an

imaging cytometry assay utilizing statistical analysis of cellular

morphology. Since Rho-associated protein kinase (ROCK) [26]

inhibition of endothelial cells has been seen as a potential

therapeutic approach to reduce angiogenesis in cancer [27], we

measure the morphology of EA.hy926 (an endothelial cell line

[28,29] which possess vascular formation potential in an ECM gel)

in response to ROCK inhibition by H1152. The dose-dependent

morphology of endothelial cells was quantitatively determined

using shape parameters.

Materials and Methods

Material preparation and nomenclatureThe NFES solution is based on an aqueous mixture of gelatin

(porcine skin Sigma-Aldrich), acetic acid and ethyl acetate (Fisher

Chemicals). All these materials were used without any further

purification. Twelve different formulations of gelatin solutions

were used in this work, as detailed in Table 1 in Results and

Discussion. The appellation of each solution will be designated

according to the following convention. The first part, with a prefix

G, represents the weight percentage of gelatin; the second part,

with a prefix A, represents the total weight percentage of acid and

its derivative (which are mixtures of acetic acid and ethyl acetate);

finally, the third part specifies the ratio of acetic acid to ethyl

acetate in a bracket. For example, G8-A72(1.5) indicates a solution

containing 8wt% gelatin and 72wt% acid-acyl additives, while the

ratio between acetic acid to ethyl acetate is 3:2. We also adopt the

abbreviations of A.A. for acetic acid, and E.A. for ethyl acetate. To

prepare the solution for NFES, gelatin was dissolved in solvents of

deionized water, acetic acid and ethyl acetate at appropriate

weight fractions, stirred at room temperature for 24 hours before

use as a homogeneous mixture.

PDMS (polydimethylsiloxane) films were used as substrates for

gelatin pattern deposition where cell culturing was performed.

PDMS (Sylgard 184, Dow Corning) was mixed at a 1:10 hardener-

to-resin ratio, degassed, and poured over a silicon wafer. It was

then spin-coated (Electronic Micro Systems LTD, Model 4000) at

200 rpm for 120 s, and crosslinked at 60oC for 3 hrs before

peeling off. The PDMS substrate was measured of 660 mm in

thickness. Prior to NFES, plasma treatment was carried out to

impart hydrophilicity to the PDMS surface (Femto Science, 30 s,

25 sccm, power 10). Bovine serum albumin (BSA), purchased from

Sigma Aldrich, was dissolved in distilled water to give a

concentration of 0.2% w/v. 20 mL of the solution per centimeter

squared was used to coat the plasma-treated PDMS. The solution

on the PDMS was then left to evaporate.

Fabrication of a gelatin array for cellular studiesThe experimental scheme of NFES is shown in Fig. 1. The set-

up included a syringe pump (World Precision Instruments, AL-

1000), a 1 ml syringe (BD Plastipak), a needle tip (BD Microlance,

19G), a high voltage power supply (Stanford Research Systems,

INC., PS350/5000V-25W), an X-Y motion stage (PI micos, LMS-

60), and a Z motion stage (Thorlabs, L490MZ/M). The syringe

needle was ground blunt and its outer diameter was 1.08 mm. The

anode of the high voltage power supply was attached to the syringe

needle, and the cathode was connected to the grounded collector.

Silicon wafers or PDMS films were used as substrates for gelatin

deposition. The Z stage was adjusted so that the distance between

the syringe tip and the substrate surface was 1.25 mm. Prior to

patterning, a droplet of *0.2 mL gelatin solution was pushed out

of the syringe tip using the syringe pump. Subsequently, a voltage

of 1000 V was applied between the spinneret and the deposition

collector to initiate an electrified jet for pattern deposition.

The cell assay used here was constructed of a gelatin fibril array

of 40 mm spacing, patterned on 0.5 cm|0.5 cm PDMS pieces.

Crosslinking of gelatin was required to make it insoluble. After

NFES, the gelatin array was immersed in a crosslinking solution

formed by 25 mM of EDC (1-ethyl-3-(dimethyl-aminopropyl)

carbodiimide hydrochloride, Alfa Aesar) and 10 mM of NHS (N-

hydroxyl succinimide, Alfa Aesar) in ethanol/distilled water (9:1

v/v) following the procedure by Zhang et al. [30]. Crosslinking was

completed at 4oC after 24 hrs. The gelatin-PDMS construct was

then rinsed with distilled water and ethanol to remove excess

crosslinking agents and dried in a desiccator for 24 hr. Sterilization

of the construct using 70% ethanol was applied prior to cell

culturing.

CharacterizationNFES solution properties. In order to establish the corre-

lation between the fiber morphology and the solution properties,

we measured pH, conductivity, viscosity and surface tension of the

different gelatin solutions. pH was measured using a micropro-

cessor pH meter (pH 210, Hanna Instruments) at 26.8oC.

Viscosity was examined through a rheometer with a measuring

cone CP 50-2/TG (Physica MCR 501, Anton Paar) at 20.6oC. A

fixed strain of 5% was applied, while the sweep frequency changed

from the initial angular frequency of 100 rads/s to the final

angular frequency of 0.1 rads/s. The conductivity of the gelatin

solutions was determined using a conductivity probe (InLab 731

ISM, Mettler Toledo) at 28oC. Finally, the surface tension of the

gelatin solutions (at 24.9oC) was deduced from the pendant drop

method using ImageJ [31].

Physical properties of gelatin fibres. For the fiber

morphology characterization, the in-plane morphology of the

gelatin patterns was examined using confocal microscopy (Zeiss

LSM 510), and scanning electron microscopy (SEM, XL 30 sFEG,

Philips). For SEM, samples were gold coated to improve charge

dissipation during imaging. To identify the local profile of a dried

fibre (i.e. the shape of an individual stripe), atomic force

microscopy (Veeco Dimension 3100, Digital Instruments) was

used, with imaging performed at tapping mode to minimize

surface damage.

The topology and the mechanical properties of the fibre in

liquid were further characterized using in-situ atomic force

microscopy (AFM) imaging and indentation based on a Nanowi-

zard AFM (JPK Instruments, Germany). All measurements were

taken in distilled water at room temperature. Preparation

procedure involved fixing the sample onto a piece of mica using

Araldite and inserting it into a liquid cell. The samples were pre-

soaked in distilled water for over 10 min prior to measurement. In

1-D Collagenous Topography Patterns for Image Cytometry


order to match the stiffness of the substrate and optimize

sensitivity, a relatively soft cantilever was used. A silicon tip

(NSC36, Mikromasch) was chosen with a cone half-angle of 15o.

The tip spring constant was calibrated in water using indentation

on mica and thermal activation. This approach gave a tip

sensitivity of 28+2 nm/V and a normal spring constant of

2.0+0.2 N/m. In-situ AFM imaging was performed in the

intermitted contact mode (also known as tapping mode) and the

force mapping was taken in the contact mode. In the force

mapping measurement, the tip approached the sample with a

constant speed. When the tip reached the surface, it indented the

substrate until a pre-set maximum load was reached. This ensured

that the indentation was controlled to be less than 100 nm which is

*10% of the fibril thickness and should be small enough to not

damage the sample material. 32|32 px2 indentation curves were

collected on a square area. The size of the area was

10 mm610 mm which gave a resolution better than

300 nm|300 nm. Having adapted the Hertzian model (JPK

Table 1. Compositions of gelatin solutions used for NFES patterning.

Notation Gelatin, wt% Water, wt%Acetic acid,wt%

Ethyl acetate,wt% A.A.:E.A. Morphology

G8-A72(1.5) 8 20 43.2 28.8 1.5 none

G10-A70(1.5) 10 20 42 28 1.5 beaded

G10-A70(1) 10 20 35 35 1 none

G10-A70(4) 10 20 56 14 4 beaded

G11-A69(1.5) 11 20 41.4 27.6 1.5 beaded

G11-A64(1.5) 11 25 38.4 25.6 1.5 beaded

G11-A59(1.5) 11 30 35.4 23.6 1.5 straight

G11-A54(1.5) 11 35 32.4 21.6 1.5 straight

G11-A49(1.5) 11 40 29.4 19.6 1.5 curly

G11-A69(2) 11 40 46 23 2 beaded

G12-A68(1.5) 12 20 40.8 27.2 1.5 beaded&straight

G15-A60(1.5) 15 25 36 24 1.5 straight

The compositions of the gelatin solutions are detailed, with the global morphology of the pattern resulted for each composition also indicated.doi:10.1371/journal.pone.0093590.t001

Figure 1. Versatile gelatin fibril patterns can be formed by near-field electrospinning over a large area. (a) Scheme of the NFESpatterning process. (b) A 2 inch PDMS sheet containing arrays of gelatin patterns. (c)–(e) Confocal images of fibril patterns with bead-on-strings, coils,and uniform straight fibrils respectively. (f)–(h) Scanning electron micrographs showing typical regions of the fibril patterns illustrated in (c)–(e).doi:10.1371/journal.pone.0093590.g001



Data Processor) [32], which was developed from the Oliver and

Pharr model [33], analysis of the force curves provided the spring

constant and Young’s modulus of the substrate.

Chemical properties of gelatin fibres. Fourier transform

infrared spectroscopy (FTIR) (Thermo Scientific) was used to

analyze the absorption spectrum of gelatin samples, that is,

uncrosslinked gelatin film, crosslinked gelatin film, uncrosslinked

NFES gelatin fibrils, crosslinked NFES gelatin fibrils, and finally

gelatin pellets compacted from mixtures of KBr and as-received

gelatin powder. The collected absorption spectra were converted

to transmission spectra using the Omnic program. The baselines

were adjusted to allow comparison of the spectra after smoothing.

To evaluate whether the incubation of cells with crosslinked

gelatin would alter its surface chemistry, attenuated total

reflectance (ATR) infrared spectroscopy (Bruker Tensor 27

Spectrometer) was performed. Gelatin films were made by leaving

drops of gelatin solutions of electrospinning grade on PDMS

substrate to dry in the fume cupboard at room temperature for

24 hr. They were cross-linked according to the same procedure

above for 24 hrs. The sides were flipped and cross-linked for

another 24 hr to ensure homogeneous cross-linking across the film

thickness. The films are then rinsed with deionised water and

ethanol before storage in ethanol. For cell culturing, the films were

washed in PBS, and then soaked in the cell culture medium for

30 min. Cells were directly seeded onto the gelatin film at a 1=2confluency and left to grow for 18 hrs (see culture conditions

below). Subsequently, the cells were detached from the surface by

trypsinization. After soaking the cell-detached film in deionised

water overnight to remove any attached proteins, the films were

then soaked in ethanol for 2 hr before drying in the vacuum oven

for 1 hr. The samples were then taken for ATR scanning focusing

on the surface where cells had resided. In addition to the cell-

incubated samples, reference film samples subjected to the same

solution incubation process without cell seeding were also studied.

Endothelial cell assayHuman umbilical vein cell line EA.hy926 (American Type

Culture Collection, Manassas, VA; passage 10–20) was main-

tained in Dulbecco’s modified eagle medium (DMEM, Gibco)

supplemented with 10% fetal bovine serum (Sigma) and 1%

penicillin-streptomycin (Sigma) in 5%CO2 at 37oC.

When an assay was conducted, cells were seeded to obtain a

coverage of 100 cells/mm2 on gelatin patterned PDMS, or bare

PDMS (as a reference). After seeding, cells were allowed to attach

and interact with the substrate for the first 12 hrs in normal

culture medium. Subsequently, the media of selective samples

were replaced with ROCK inhibitor-containing media (at an

H1152 concentration ranging between 0.5 to 50 mM). Incubation

of another 8 hrs took place before the cell fixation. In parallel,

untreated cells were maintained in normal culture medium for the

entire 20 hr period before fixation.

For immunostaining, cells were fixed with 4% of paraformal-

dehyde solution in PBS (phosphate buffer saline) for 20 min. After

being washed twice with buffer solution (0.05% Tween-20 in PBS),

the cells were permeabilized with 0.1% Triton X-100 solution in

PBS for 5 min. Then the sample was washed twice with the buffer

again and the blocking solution (1% BSA in PBS) was applied for

30 min. Afterwards, the sample was incubated with the anti-

Vinculin primary antibody (purchased from Invitrogen, used with

1:400 dilution in blocking solution) for 1 hr at room temperature.

The sample was washed three times (5 min for each) with the

buffer solution. Finally, the sample was incubated for 50 min in a

PBS solution containing Phalloidin (1:400 dilution), Vinculin

antibody (Invitrogen, 1:400 dilution) and Hoechst (Invitrogen,

1/10000 dilution). The sample was then washed three times

(5 min for each) with the buffer solution. Images were acquired

using a Leica confocal microscope (TCS SP5 II) at randomly

chosen areas of a sample, within 3 days after staining.

Quantitative analysis of cell morphologyMorphology analysis was conducted for the fixed cell samples

based on immunofluorescence images. The morphological pa-

rameters were calculated using the ‘‘Measure Object Size and

Shape’’ module in CellProfiler software (Broad Institute, USA)

[34,35]. The cell orientation was calculated by fitting an ellipse to

the cell body, so that a net cell alignment angle was obtained by

measuring the angle between the cell’s major axis and the fibril.

We found that the software was able to correctly identify and

segment both isolated and connected cells. It was able to compute

the cell areas with reasonable accuracy. For example, the

computed cell area for the untreated cells on bare PDMS was

*600 mm2 for most of the population, which corresponded to

cells of *28 mm in diameter. This compared well with the manual

measurement of these approximately circular attached cells. The

perimeter estimation was on the other hand less accurate and

subject to underestimation since it heavily relied on the visibility of

the cell edges when measured using fluorescence. Fine cellular

protrusions with widths of 1 mm or below were not expected to be

accounted for, since our imaging condition was limited by 1 pixel

(0.51 mm). Finally, it is noted that we did not exclude the small

number of artifacts which apparently led to results of reduction in

cell areas since they did not affect the overall statistical analysis.

Results and Discussion

Rapid single-step patterningFor NFES of gelatin, a static droplet of gelatin solution was

utilized to generate a continuous jet string for pattern deposition,

see Fig. 1(a). Once electrified, the static droplet carried positive

charges on its outer surface. At the bottom of the semi-sphere,

where the electric field was at its greatest strength, surface

cohesion was broken at a sufficient voltage level, and a polymer jet

was ejected, neutralizing upon coming into contact with the

collector. During continuous fabrication, a current reading of

about 0.005 mA was gauged. By programming the X-Y motion

stage in a regular fashion, the encroaching jet can be made to

produce tailored patterns. No initiation methods such as tip

probing [36] were implemented, because the edge effects of the

collector produced a non-uniform and time-dependent electric

field which rearranged the charge distribution and this led to

shape distortion that provided the activation energy. No contin-

uous flow [17] was used due to the precision control of the

electrospinning parameters. The weight of the deposited fibril was

insignificant in contrast to that of the droplet (*0.5% for one run),

which justified the use of a static droplet. There are three benefits

associated with such an ECM patterning technique. First, direct

fabrication on a soft, insulating gel-like substrate is facilitated.

Secondly, it presents an expeditious protocol for patterning over

large areas with low material usage. It only required a single step

of *15 min to pattern a 2 inch wafer with a line density of 20

lines/mm. This incorporates much less processing steps than other

existing template-based methods such as stamping or photo-

patterning. Thirdly, by virtue of the versatility of electrospinning,

other ECM fibrils are possible, such as elastin and laminin. An

example of the gelatin-patterned PDMS is shown in Fig. 1(b).

In the absence of disconnected polymer clusters, often produced

by electrospraying process [37], three distinct types of in-plane,

global morphological features are displayed in this NFES work,



that is bead-on-strings, coils and straight fibrils. Their optical

images and scanning electron micrographs are shown in Fig. 1(c)

to (e), and Fig. 1(f) to (h) respectively. It is noted that, only specific

solution combinations can permit successful NFES. Solution

properties tightly control both the in-plane fibril topography,

and the local scale topology features. We will now discuss these

effects below separately.

Parameter control for global patterns. Table 1 summa-

rizes the compositions of various gelatin solutions used in this

study, as well as their resulting pattern morphology. As stated in

the Materials and Methods section, the nomenclature of G(gela-

tin)wt%-A(acid-acyl)wt%-ratio is used. Analysis of the composition

and patterning ability indicates that the total content of acid-acyl is

the dominating factor in determining patterning ability. As the

acid-acyl content increased beyond *60wt%, no fibrils were

formed. The change in gelatin concentration also affected the

solution characteristics such as conductivity, pH, surface tension

and viscosity to different extents (see Fig. 2 and Fig. 3). These

solution characteristics have been known to affect the performance

of conventional far-field electrospinning. Here, we establish some

key solution indicators for generic NFES patterning of an aqueous

solution of a biopolymer.

First, by analyzing the different solution properties vs patterning

abilities, we suggest that conductivity can be considered as the

most important indicator for global morphology control (under a

fixed hardware configuration). The dependence of conductivity on

the acid-acyl content, i.e. (A.A.zE.A.), is shown in Fig. 2(a). In

general, the electrolytic conductivity decreases with the increase in

(A.A.zE.A.) percentage within the range of our studies. In

particular, the feasibility of NFES and the three regimes of

patterns can be well defined by the conductivity. NFES process

would not be initiated until the solution conductivity had reached

*0:5 mS/cm, with the onset of beaded structures afterwards.

Above *1 mS/cm, curly fibers formed. Hence straight fibrils

were only produced within the conductivity range of 0.75 to

1 mS/cm. Now returning our focus to the solution composition, it

is important to note that, both gelatin and ethyl acetate have

molecular structures, whereas acetic acid has a partially ionic

structure; thus acetic acid (A.A.) is the sole electrolyte out of the

three solutes [38]. The electrolytic conductivity of A.A. in water is

not a monotonic function of concentration. The conductivity of

acetic acid only aqueous solution peaks at around 25 wt% following

Kohlausch’s Law, and subsequently decreases as the high

concentration effect becomes prominent owing to more inter-

ionic interaction [39,40]. For mixed electrolytes, such as our

systems studied here, the conductivity is approximately an

arithmetic mean in terms of its constituent conductivities. Hence,

given the much lower conductivity value of ethyl acetate

(*0.046 mS/cm) compared to acetic acid (*1 mS/cm) solution,

the conductivity of the solution mixture approaches that of

aqueous acetic acid. Thus, for a constant A.A.:E.A. ratio,

increasing (A.A.zE.A.) decreases the solution conductivity. This

gives rise to the general decreasing conductivity trend shown in

Fig. 2(a). On the other hand, when the (A.A.zE.A.) was kept

constant, the solution conductivity should increase when the E.A.

weighting is decreased (i.e. reduction in the poorly-conductive

solvent). Therefore, for the three points associated with G10-A70,

an increase in the A.A.:E.A. ratio from 1, to 1.5 to 4 had increased

the conductivity across the threshold for jet initiation, and beaded

fibril structures resulted. On the other hand, we observe in Fig. 2(a)

that the variation in gelatin content from 8wt% (G8) to 15wt%

(G15) negligibly changed the conductivity trend. In particular,

both G11-A59(1.5) and G15-A60(1.5) produced straight fibrils.

Next, we focus on the dependence of fibril morphology on pH.

The pH measurements for all the gelatin solutions are shown in

Fig. 2(b). It seems that pH does not play an indicative role in the

NFES morphology. A solution pH of *2:6 had entered all the

three regimes of curly, straight and beaded fibers; though for

pHv2:58, the solution resulted in either beaded fibrils or no

jetting. Given the small equilibrium constant (Keq~2:25|10{2)

for the proton dissociation of A.A. [41], the pH value is solely

determined by the A.A. mixing content. This explains the large

depression in pH for G10-A70(4) (with a beaded structure at

pHv2), which had an A.A. content of 55wt%, much above the

others.

We will next consider the correlation between surface tension (at

the liquid/air interface) and fibril morphology, based on Fig. 2(c).

The addition of A.A. (surface tension*27.6 mN/m) and E.A.

(surface tension*23.9 mN/m) dramatically lowered the surface

tension of the gelatin solution (cf. that pure water has a surface

tension of *72:0 mN/m). This is in accordance with an equation

proposed by Szyszkowski [42]. A trend of decreased surface

tension with increased (A.A.zE.A.) content was first observed, but

a transition in behavior took place for (A.AzE.A) content

Figure 2. Solution properties controls global fibril morphology. Different solution properties are plotted against the total acid-acyl contents(A.A.zE.A.) for (a) conductivity, (b) pH, and (c) surface tension. The approximate regimes of fibril morphology are highlighted in each graph. For (b)and (c), a pair of blue lines are drawn to enclose the range of conditions which resulted in the formation of straight fibers. It is to note that for G10-A70, the A.A.:E.A. ratios of 1, 1.5 and 4 were tested, and this can be seen as the increase in conductivity in (a).doi:10.1371/journal.pone.0093590.g002



w65wt%. Like pH, the surface tension does not seem to act as a

good indicator for morphology control, since for example, a

surface tension between 31 mN/m and 33 mN/m for a straight

fiber morphology also overlapped the surface tension accompa-

nying the no jetting regime. However, it is interesting to note that,

fibers of beaded morphology were all produced by solutions of low

surface tension, i.e. below *31 mN/m.

During electrospinning, the initiation of a polymer jet depends

on the counteracting actions of charge repulsion and surface

tension. Under a fixed electric field, the repulsive force generated

depends on the number of net charges present on the surface, a

factor which can be regulated by the solution conductivity.

Excessive conductivity results in whipping instability (curly fibrils),

whereas low surface tension leads to beading. Comparing the

results of Fig. 2(a) and (c), we see that the conductivity is more

strongly affected by the (A.A.zE.A.) content than the surface

tension in our range of studies. In other words, tuning the solution

conductivity may be regarded as a good strategy to obtain stable

jetting for NFES patterning. Since the gelatin solubility depends

on the A.A. content, a balance between solubility and conductivity

should also be sought.

Parameter control for local topography. Here, we inves-

tigate the local topography resulting from NFES patterning of

gelatin fibrils. For the purpose of precision patterning, our

discussion will focus on straight fibrils, by measuring their widths

and cross-sectional heights. As discussed previously, straight fibrils

could be produced by solution G11-A54(1.5), which had a

conductivity of 0.98 mS/cm and surface tension of 33 mN/m.

To unbiasedly evaluate the pattern uniformity, the widths of the

dry fibrils at regular intervals were measured (shown in Fig. 3(a)

top panel), and the widths at randomly chosen positions across

different dry fibrils were also determined (shown in Fig. 3(a)

bottom panel). Both width statistics indicate an average dry fibril

width of *2:5 mm, under the fixed hardware condition as stated

in Materials and Methods. The distribution of widths within a single

fibril is narrower than that of the cross-fibril widths. Nevertheless,

more than 80% of the fibrils have dry widths within the 2–3 mm

interval. It is also expected that further optimization in the

fabrication condition may reduce the variance in widths across

different fibrils.

We do not observe significant variation in the average fibril

width with respect to the gelatin concentration. However, the

height of the fibril was significantly altered. Straight fibrils were

mostly cast into a smooth parabolic silhouette, and the height

stayed relatively constant along the fibril. A parabolic profile

rather than a circular one might imply incomplete solvent

evaporation as the jet reached the target substrate. Under this

condition, solution viscosity was found to be the major factor

determining the fibril height. The viscosity findings for all the

gelatin solutions are plotted in Fig. 3(b). It is known that in an

acidic regime, the viscosity of gelatin solution is approximately a

linear function of the gelatin concentration. However, with the

multiple solution components incorporated here, the gelatin

concentration did not strongly affect the viscosity from 8 wt% to

12 wt%. In fact G(8) to G(12) samples had their viscosity values

scattered around 0.1 Pa:s. For a 15 wt% sample G(15), a

significant increase in viscosity to 0.4 Pa:s was observed. In

particular, solutions G(8) to G(12) all showed a Newtonian flow

characteristic, while G(15) displayed reduced viscosity with

increasing rate of shear (a typical shear thinning behavior). With

an increased viscosity, G(15)-A60(1.5) resulted in an aspect ratio

(i.e. height to diameter) of *1 : 3, much greater than the aspect

ratio of *1 : 10 achieved by G(11)-A59(1.5), as shown in

Fig. 3(c)&(d). It is noted that the high solution viscosity complicates

the electrospinning process by prolonging the equilibrium

settlement of the droplet into a fixed volume, during which a

reduction in volume through evaporation can occur, in violation of

precision control. The degree of hydration [43] of gelatin in

relation to the high viscosity can be determined, with a

strengthened effect in the acidic environment, in conjunction with

its molecular weight and geometry [44]. Moreover, besides its

subtle influence on the topology of the patterned fibrils, viscosity

may also govern the mechanical properties of the resulting fibrils.

This stress in viscosity over concentration has been reported

recently [45].Chemical and physical characteristics of NFES

fibres. The pH range for the NFES gelatin solutions was from

2.01 to 2.68. Gioffre et al. [46] have performed wide-angle X-ray

diffraction analysis and demonstrated only partial denaturation of

the triple-helix content of gelatin in this pH range for its integrated

intensity of the 1.1-nm reflection. FTIR spectra are shown in

Fig. 4(a) to evaluate the effects of NFES and crosslinking on the

chemical structures of gelatin. The spectra show mostly similar

peak characteristics originally present in the as-received gelatin

powder [47]. The peaks that are present in all spectra include the

Figure 3. Local topology of gelatin fibril. (a) Histograms of width statistics sampled at regular intervals of a single fibril (upper panel, n~272),and randomly chosen positions across different fibrils (lower panel, n~141). (b) Plot of viscosity against the total acid-acyl contents (A.A.zE.A.).(c)&(d) Scanning electron micrographs showing the freeze-fractured cross-section produced from solutions G15-A60(1.5), and G11-A59(1.5)respectively; their respective viscosity is indicated by an arrow in (b).doi:10.1371/journal.pone.0093590.g003



amide A band (N-H stretching mode) detected at around

3320 cm21, the amide I band (C~O stretching mode) at around

1650 cm21, the amide II band (N-H bending mode) at around

1550 cm21, the methyl bend or scissoring band at around

1450 cm21, the methyl rock at around 1330 cm21, the carboxyl

peak (C-O stretching) overlapping the amide peak (C-N stretching)

at around 1250 cm21, and finally the alcohol peak (C-O

stretching) at around 1080 cm21 [48,49]. For the gelatin subjected

to crosslinking, we notice that the peak at 1050 cm21, which

belongs to ester (C-O stretching), has become more distinctive.

This suggests the esterification reaction [50] between the

carboxylic group and the alcohol group had taken place during

the crosslinking. ATR infrared spectroscopy of the cell-incubated

film surface showed unnoticeable change in the peak position or

the relative peak height compared to the untreated film. Thus we

suggest that the surface chemical composition of the crosslinked

gelatin (as measured by a penetration depth of *1 mm based on

typical settings of ATR), had remained similar in compositions to

its original form over a period of 18 hr cell incubation. However,

we do not exclude that changes to the top tens of nanometer

surface had taken place. Further studies using techniques such as

XPS [51] can elucidate this.

The stability of fibre geometry was also investigated by atomic

force microscopy (AFM). It has been found that the crosslinking

process reduced the fibril height by *20%, but left the fibril width

unchanged. Analyzing the effect of hydration on the structural

integrity of the fibers, we found that the crosslinked fibril was able

to retain its shape after it had been soaked for 24 hrs in deionised

water and dried (see Fig. 4(b)&(c)). Furthermore, the physical

property of the hydrated gelatine fibril was investigated using in-

situ AFM as a reference to the physical response experienced by

ECs. To interpret the force-displacement results using the

Hertzian model, the following assumptions were made. (1) The

indenter was rigid compared to the sample material; (2) During

loading, the substrate might be deformed plastically as well as

elastically. However, only the elastic component contributed to

unloading. Therefore analysis was performed based on the

retraction curves; (3) The material was homogeneous and isotropic

and the Young’s modulus was independent of the strain state; (4)

The tip of the cantilever was narrow compared to the curvature of

the fibril. During indentation, the fibril surface was roughly flat

and loading direction was perpendicular to the surface. The

Poisson’s ratios of both gelatine and PDMS were assumed to be

0.5 as reported by Richards and Mark [52]. As both gelatine and

PDMS are viscoelastic materials, the Poisson’s ratio is highly time-

dependent and can vary under different stress states [53]. In our

experiment, each indentation was performed within the time scale

of seconds. Thus, it can be assumed that the Poisson’s ratio

remained constant under such fast deformation.

Figure 4. Physical and chemical properties of gelatin fibrils. (a) FTIR spectra of as-received gelatin powder (P), a cast gelatin film (F), thecrosslinked gelatin film (Fx), NFES gelatin fibrils (NF), crosslinked NFES gelatin fibrils (NFx), and crosslinked gelatin films post cell culturing for 18 hrs(Fx(C18)). (b) AFM profiles of a crosslinked gelatin fibril before being immersed in water (left), and after being immersed in water for 24 hrs and dried(right). (c) Cross-sectional profile of the fibrils presented in (b). (d) In-situ AFM imaging of a hydrated gelatin fibril. (e) Young’s modulus mapping of acentral fibril region. (f) Histogram showing the range of Young’s modulus values for the gelatin fibril and PDMS respectively.doi:10.1371/journal.pone.0093590.g004



In the AFM image of Fig. 4(d), the hydrated fibril is shown with

a height of *1 mm and a width of *4 mm. The increase in fibril

dimension from a height to diameter ratio of v1 : 10 to *1 : 4,

indicates the swelling of gelatine in water. In addition, the poorly

defined boundary of the fibril suggests that the wet gelatine fibril

has become considerably soft. This behavior is further confirmed

in the Young’s modulus map (Fig. 4(e)). Qualitatively, the distinct

dark/bright region shows that the gelatine fibril exhibits a lower

Young’s modulus than the PDMS. Only the central part of the

fibril has been considered for quantitative analysis. This eliminates

points where the direction of indentation was not normal to the

surface as well as where slipping of the tip occurred. The average

Young’s modulus of fibril was measured to be 226+35 kPa and

that of the PDMS was 1+0.5 MPa. The uncertainty in the

Young’s modulus is up to 20%. Such deviation is contributed by

both the sensitivity and spring constant of the cantilever tip. In

addition, adhesion of the substrate, especially gelatine, can cause

over-estimation of the contact area [54]. The standard deviation of

the PDMS modulus is greater than that of the gelatine fibril. This

can be caused by surface-bound BSA which can be seen in the

background of Fig. 4(d). Furthermore, it is important to notice that

the Young’s modulus distribution (Fig. 4(f)) is approximately

Poisson. This proves the results are statistically validated. Finally,

the spring constant of the sample material was evaluated by

analyzing the initial gradient of indentation curves. The indenta-

tion force curves show that the resultant spring constant of gelatine

is 0.080+0.006 N/m and that of PDMS is 0.24+0.03 N/m. The

uncertainty is approximately 10% which agrees with the

uncertainty of the tip spring constant. Both spring constants are

at least one order of magnitude smaller than that of the tip. This

validates assumption(1) stated previously.

Topography-guided endothelial patternsModulation of cellular morphology towards a cord-like

phenotype. Endothelial cell line (EC) EA.hy926, known to

display a number of features characteristic of vascular ECs [28],

was interfaced with NFES gelatin arrays. A parallel fibril array is

chosen since it is the most basic mimic of the surface topography of

ECM fibrils. Benefited by the large sample area and well-defined

patterns on a transparent, 2-D substrate, cell imaging was

performed with a conventional inverted microscopic set-up, and

multiple regions were monitored at once to obtain sufficient

statistics. Figure 5(a) shows that 20 hours after plating, many of the

ECs were found to form connected cell strings of a few to over ten

cells on the fibril array. An image slice of the reconstructed 3-D

conformation of a cell string is shown in Fig. 5(b), which illustrates

the presence of actin filaments around the cell surface. Each cell

string resembles the pre-capillary cords formed in a Matrigel [29]

(a gelatinous protein mixture). However, we did not observe lumen

formation within 20 hrs culturing according to the cell string’s 3-D

structure. Nevertheless, the cord-like structure is not presented on

a featureless 2-D surface in the control sample, Fig. 5(e).

Various factors could trigger the cell attachment and the

formation of such a cell string on the gelatin fibril. The precise

mechanism does not fall in the scope of this study, though we

postulate three possible reasons. Firstly, gelatin extracted from skin

was used in this study, and collagen type-I was known to be the

major composition of skin. Extensive studies have showed collagen

type-I to initiate the angiogenesis process after the degradation of

basement membrane layer (see studies summarized in a compre-

hensive review [55]), thus the gelatin fibril here could exhibit

similar ligation sites to the EC integrins [55]. Secondly, the quasi

1-D topography of the fibril is expected to play a role in providing

the physical constraints, and thus the initial cell polarization which

is required for an angiogenesis process. Previous studies have also

demonstrated a critical role of the adhesion topography in

controlling mono-layer or cord-like morphogenesis of ECs [56].

Finally, recent studies also point to the importance of the substrate

mechanical properties in controlling cell decision [57]. As shown

in our mechanical measurement by in-situ AFM, the PDMS

exhibits a modulus of *1 MPa, which is five times to that of the

hydrated gelatin fibrils (*200 kPa). However, it is to note that

previous work [56] which showed cord-like EC morphology, was

based on patterning a thin layer of adhesion molecules on PDMS;

thus the cells would have directly ‘‘felt’’ the stiffness of the PDMS

interface. With the above, we can not conclude whether the ECs

may prefer a more mechanically compliant substrate provided by

the gelatin. Further studies in manipulating the mechanical

mismatch between the fibril and the base layer may be of great

interest.

To illustrate the application of such a cell assay, we investigate

the morphology changes of ECs subjected to different treatments,

i.e. normal cells or ROCK-inhibited cells cultured either on a

gelatin array or a PDMS reference (see Materials and Methods for

details). In various cell types, ROCK-dependent regulation of

actin cytoskeleton enables cells’ adaption to variation in the

physical properties of the surrounding environment [26,58]. Of

particular interest to ECs and potential medical implications,

ROCK inhibition is seen as a cancer treatment strategy to reduce

angiogenesis [27]. ROCK inhibition was found to hinder the tube

formation processed in tumor-derived ECs [59] and early-stage

VEGF-mediated vascular formation [27] in 3-D gel; however,

studies by Bryan et al. [27] noted that the inhibition did not affect

the maintenance of an established vascular network. Since studies

performed previously had shown that the morphological re-

arrangement of EA.hy926 towards a capillary-like structure

occurred within 12 to 16 hrs after seeding in a Matrigel [29],

and a similar onset was also found on our gelatin-fibril array,

ROCK inhibition by H1152 was implemented 12 hr after plating.

By doing so, we suggest the experimental results are more closely

related to the effect of ROCK inhibition on the capillary network

forming process. To investigate the concentration effect of the

inhibitor, H1152 was added at a concentration ranging from 0.5 to

50 mM. We use the notation of I(concentration), where I(5 mM), for

example, implies an inhibitor concentration of 5 mM.

Immunofluorescence images of the normal and I(5 mM) ECs are

compared in Fig. 5(c) to (f). With I(5 mM), we observe an apparent

increased area of the cell body (comparing Fig. 5(d)&(f) to (c)&(e)).

The slender membrane protrusions were also much longer than

the untreated cells. ROCK inhibition has been shown to promote

the formation of cellular projections in a number of cell types

including ECs [27]; thus our observation is in accordance with the

literature report. In particular, for the fibril-interfaced, H1152

treated sample, the protrusion extension was most dramatic for the

projection guided along the fibril. This dominating protrusion can

reach a length over 200 mm. The long protrusions prevented close

packing of neighboring ECs along the fibril, such that tightly

connected cell strings are not observed. Therefore, under H1152

treatment, the ECs had lost the morphological phenotype of a

capillary partially exhibited by untreated, fibril-interfaced ECs.

To quantitatively assess the observed difference described

above, we utilize an open source software CellProfiler [34,35] to

segment and extrapolate the cellular morphological parameters.

Combining the large, regular patterns of gelatin fibrils, and the

highly adaptable image analysis procedure, a large number of cells

can be analyzed from which the information of a single cell is

extracted. Examples of the segmentation results are shown in

Fig. 5(g)&(h). The number of morphological parameters that



resulted are extensive. We choose first to investigate the

correlation between the cell perimeter and cell orientation because

a visible protrusion may significantly increase the cell perimeter

without dramatically changing the cell area. Cell orientation is

used in order to evaluate ECs’ sensitivity to the geometrical/

biophysical cues presented by the fibrils. Cell area is also chosen

since it is one of the most commonly investigated morphological

parameters, and it can also be used to validate the accuracy of

segmentation (see Materials and Methods). We will now discuss these

results in details below.

Image cytometry revealing interface-dependent response

to ROCK inhibition. Figure 6 illustrates the scatter plots of the

cell perimeter vs cell orientation, for the four experimental

conditions studied here. First, by comparing the histograms of

the perimeter, Fig. 6(c),(f),(g)&(j), it is apparent that I(5 mM) has

dramatically increased the cell perimeter. The modal perimeter

(the most likely occurring perimeter) has increased from *150 mm

for the untreated cells, to *375 mm for the inhibited cells. It is

important to note that the presence of fibrils did not affect the

most probable perimeter associated with cells subjected to the

same treatment, i.e. from comparing (c)&(g) or (f)&(j); but a higher

proportion of cells exhibited increased perimeters. In other words,

discounting the effect of ROCK inhibition, cell polarization may

be the main factor contributing to additional cell perimeters.

Secondly, comparing the histograms of the orientation,

Fig. 6(a)&(b) vs (k),&(l), we find that the gelatin array had induced

EC alignment with respect to the fibril axis. For untreated ECs,

*50% of the cells had their major axes within +10o from the

fibril axis, and this was comparable to the I(5 mM) counterpart

with *40% of the population. Finally, scrutinizing the scatter

plots of Fig. 6(e)&(i), we observe that the extremely large perimeter

values are centered around the 0o orientation in Fig. 6(e).

To evaluate the H1152 concentration dependence of the EC

morphology, similar scatter plots of the cell perimeter vs. cell

orientation were also produced for other treatments shown in

Fig. 7(a–d). One clearly observes an increasingly wider distribution

of the scatter plot w.r.t H1152 concentration. This large deviation,

as a signature of the cell’s reduced ability to control its shape,

confirms the literature reports [58,60]. Moreover, the cell

perimeter vs. the cell area was also plotted which gives information

on the cell’s strategy to adapt its shape to a substratum. As shown

in Fig. 7(e–h), their dependence was mostly confined between two

boundaries, i.e. slopes of 0:15 mm21 (lower bound) and 0:6 mm21

(upper bound). It also seems that the cell perimeter vs. cell area

dependence moves from the lower bound to the upper bound with

increasing ROCK inhibition. However, ECs’ ability to sense the

insoluble biochemical factors (i.e. presented by the gelatin fibrils)

was still retained. This is based on the fact that the cell orientation

is still centered at 0o in Fig. 7(a–d) for a H1152 concentration up

to 50 mM. If the cells were to lose its ability to follow the fibrils, the

scatter plot would have reversed to the form similar to that of the

reference. It is also important to note that such a cellular sensitivity

towards biophysical cues can only be probed by a fibril-interface,

not a conventional 2-D assay. In comparison with a 3-D matrix,

the unique benefit of the NFES fibrillar array lies in the ease of

microscopy and image analysis, such that quantitative study of

morphology phenotypes can be performed at a single-cell level.

To further illustrate the potential use of such a fibril assay, we

adopt two indicators to reflect the H1152 drug effectiveness. These

are the shape indicator of ‘‘Solidity’’ to reflect the protrusion

phenotype, and the sensitivity indicator of ‘‘Orientation Deviation’’ to

reflect the cells’ ability to follow the fibril’s adhesive features. Here

Solidity~As=H , where, As is the area of the shape region and His the convex hull area of the shape. Solidity is a parameter

automatically generated in the results of the ‘‘Measure Object Size

and Shape’’ module in CellProfiler, and for a solid object of

convex/round shape, Solidity~1. The rationale behind choosing

this shape parameter as an indicator is because an added

Figure 5. Modulation of endothelial cell (EC) pattern. (a) Assembly of endothelial ‘‘cell strings’’ over a large area of gelatin fibril pattern. (b) Animage slice of the reconstructed 3-D conformation of a selected cell string. (c) to (f) are immunostaining images of, untreated ECs on a gelatin array(c), ROCK inhibited ECs with I(5 mM) on a gelatin array (d), untreated ECs on bare PDMS (e), and ROCK inhibited ECs with I(5 mM) on bare PDMS (f); (g)and (h) are the false color segmentation images for (e) and (f) respectively. Color code for immunostaining: blue = Hoechst stain, green = Phalloidinstain, and red = Vinculin anti-body stain.doi:10.1371/journal.pone.0093590.g005



protrusion can be seen as incorporating a pronounced concave

outline to the cell shape, making Solidityv1. The Orientation

Deviation is the standard deviation of the orientation angle

measured from the fibril axis. For a population of randomly

oriented objects taking +90o on 2-D, the mean orientation will be

0o while the Orientation Deviation *52o by solvingÐP=2

{P=2h2dh. The

dependence of Solidity and Orientation Deviation on the H1152

concentration is shown in Fig. 8. The Solidity indicator decreases

w.r.t the inhibitor concentration, which is in accordance with

visual interpretation of the immunofluorescence images. The most

rapid Solidity decrease occurs for H1152 concentration v5 mM,

whereas further increase in inhibitor concentration only sees slow

decrease in the Solidity values. It is noted that the large standard

deviation of the Solidity values, which is especially prominent for

a high inhibitor concentration, is inherent in the shape variability

rather than being induced by uncertainty/errors. For the

Orientation Deviation, one observes that the greatest increase in

values again occurs for H1152 concentration v5 mM. Beyond

5 mM, increasing H1152 concentration insignificantly alters the

Orientation Deviation. The upper value of Orientation Deviation is *40o

compared to the random limit of 52o. Thus even under strong

H1152 inhibition treatment, the interaction between the ECs and

the substrate remains. Since the most apparent changes in the

Figure 6. Effect of ROCK inhibition studied by single cell morphological statistics. (a),(b),(k)&(l) Histograms of frequency distribution of cellorientation for untreated cells on NFES gelatin patterns (NF-0), ROCK-inhibited cells on NFES gelatin patterns (NF-I(5 mM)), untreated cells on PDMS(PDMS-0), and ROCK inhibited cells on PDMS (PDMS-I(5 mM)). (c),(f),(g)&(j) The corresponding histograms of frequency distribution of cell perimeters.(d,e,h,i) The corresponding scatter plots of the cell perimeter vs cell orientation.doi:10.1371/journal.pone.0093590.g006



shape indicators occur in the H1152 concentration v5 mM, future

studies may focus on this region for more detailed investigations.

Conclusion

This study reports a reliable method of contriving uniform

patterns of gelatin fibrils of different morphologies via near-field

electrospinning. Conductivity is identified, among the assorted

solution properties, as the most influential factor with its optimum

threshold of 0.75 mS/m to yield a regular, straight pattern of

gelatin mesh with tuneable line spacing through stage control.

FTIR peaks, which belong to ester C-O stretching, suggest that the

insoluble effect of crosslinking can be attributed to esterification of

the gelatin molecules. The Young’s modulus of the hydrated fibril

was measured to be 226+35 kPa, approximately five times softer

than that of the PDMS base. PDMS sheets with the NFES gelatin

fibrils mimicking aligned arrays of extracellular matrix fibrils were

tested with endothelium cells EA.hy926, which are found to be

promoted to form connected cell strings of capillary-like structures

on the fibrils. To quantitatively reflect their morphology, the

correlations between the cell area, cell perimeter, orientation and

solidity are studied. Based on this gelatin fibril assay, it is found

that ROCK inhibition reduced ECs’ contractility underlying

shape regulation, but did not diminish their ability to sense the

insoluble biophysical cues directed by the extending protrusions.

The fibril array is shown to present a more physiologically relevant

interface to elucidate cellular behaviors than a featureless 2-D

surface. At the same time, it facilitates much simpler standardized

image analysis processes than a 3-D matrix, thus providing a

potential materials platform for high throughput image cytometry

for applications in drug screening and pathology testing. In

addition to process simplicity and versatility, near-field electro-

spinning (NFES) patterned fibrils also produce a unique curved

fiber cross-section rather than a flat adhesion molecule layer like

stamping and photo patterning. Further investigations based on

varying solution viscosity over a wider range than in this study can

be conducted to relate cell behavior to the detailed fibril cross-

section topology variations. Tuning the mechanical properties of

fibrils by varying the crosslinking density can also provide a tool to

investigate cell response to the stiffness of single fibrils. The

method described here is believed to facilitate the patterning of a

wide range of ECM relevant protein fibrils, as well as other

pathological fibril assemblies such as amyloid fibrils.

Acknowledgments

The authors thank A. Buell and T. Knowles for in-situ AFM access and

assistance in the AFM measurement; also helpful discussion from

T. Mavrogordatos, and Y. Ji.

Figure 7. Dependence of cell morphology w.r.t. H1152 concentration. (a–d) Scatter plots of cell perimeter vs. orientation for ECs subject tono ROCK inhibition, and H1152 concentration at 2.5 mM, 25 mM, and 50 mM respectively. (e–h) Scatter plots of cell perimeter vs. area for ECs subjectedto the same treatment.doi:10.1371/journal.pone.0093590.g007



Author Contributions

Conceived and designed the experiments: NX AS YYSH. Performed the

experiments: NX XL CB ZL AP YYSH. Analyzed the data: NX XL CB

ZL AP AS YYSH. Contributed reagents/materials/analysis tools: NX XL

CB ZL AS YYSH. Wrote the paper: NX XL CB ZL AP AS YYSH.

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